We propose to develop a fully automated bench-top system that can generate tight-binding protein scaffolds called nanobodies against any target enzyme. The novelty of our approach lies in the ability to rapidly evolve these nanobodies using completely in vitro methods. We envision that the user injects a purified protein into this bench-top system and tight-binding Nbs will rapidly evolve within days. Our overall strategy is to mimic the genetic optimization themes found in nature and produce highly specific nanobody probes in an innovative animal-free system. The approach will include combining some newer, more risky technologies such as mRNA display and PCR-based DNA recombination methods as well as utilizing more established techniques like cell free protein expression and PCR amplification. The products of the system will be tight-binding polyclonal nanobodies against the target enzyme and the corresponding DNA, which can be used to generate larger quantities of monoclonal nanobodies. The Nbs can be directly used in everyday laboratory applications currently accomplished with traditional polyclonal antibodies. We envision that the rapid discovery, optimization, and production of nanobodies at the bench will have the same revolutionary impact that polymerase chain reaction (PCR) machines had on molecular biology decades ago. This innovative method will allow the broad biomedical research community to quickly generate highly specific biological probes, drugs, and other molecular tools for basic scientific research while circumventing the economic and time costs currently associated with the use of animals to generate antibodies. We propose to develop a fully in vitro bench-top system to create nanobodies de novo, allowing the scientific community to rapidly generate medicines, biological probes, and molecular tools for research in their own labs. The proposed idea would not require the use of animals and can be accomplished in at least one order of magnitude faster than today's in vivo methods. PUBLIC HEALTH RELEVANCE: We propose to develop a fully in vitro bench-top system to create nanobodies de novo, allowing the scientific community to rapidly generate medicines, biological probes, and molecular tools for research in their own labs. The proposed idea would not require the use of animals and can be accomplished in at least one order of magnitude faster than today's in vivo methods.